Discovery of a potent and long-acting bronchorelaxing capsazepinoid, RESPIR 4-95

BACKGROUND: Current drugs including beta-agonists have limited smooth muscle relaxant effects on human small airways. Yet this is a major site of obstruction in asthma and chronic obstructive pulmonary disease (COPD). OBJECTIVE: This study explores human small airway relaxant effects of RESPIR 4-95, a novel chemical analogue (capsazepinoid) to capsazepine. Capsazepine was recently shown to relax small airways in a way which was independent of its TRPV1 antagonism and independent of current bronchodilator drug mechanisms. METHOD: In vitro preparations of human small airways, 0.5-1.5mm in diameter and responding with reproducible contractions to leukotriene D4 (LTD4) for 12h, were used. RESULTS: RESPIR 4-95 reversibly prevented LTD4-induced contractions as well as relaxed the established tonic contraction by LTD4. RESPIR 4-95 exhibited marked improvements over the reference capsazepinoid, capsazepine, by being 10 times more potent, exhibiting twice as long duration of action after wash-out (9h), and inhibiting equally well LTD4-, histamine-, prostaglandin D2 (PGD2)-, and acetylcholine (ACh)-induced contractions. RESPIR 4-95 was distinguished from l-type calcium channel antagonist nifedipine by its greater efficacy and potency and by exhibiting increased relaxant effect by repeated exposures. Furthermore, RESPIR 4-95 was more efficacious and longer acting than the long-acting beta-agonist formoterol. CONCLUSION: Efficacy, potency, duration of action, and inexhaustibility of its relaxation of human small airways make RESPIR 4-95 an interesting lead compound for further developments aiming at drug treatment of small airway obstruction in asthma and COPD. Further work is warranted to unveil the molecular biology behind its relaxant actions.     

Discovery of 4 new cases of leprosy in a South Tunisian location

Having found a new case of leprosy from Beni Khadech, the Sfax Service of dermatology has decided to investigate in two places in this region: Elmenzla and Gattar. Beni Khadech, an endemic region, has 30 cases of leprosy in 1978 over 164 declared in Tunisia. Over 1688 systematically examined patients, 4 new cases have been discovered and 3 of which are contagious ones.     

Discovery of a CXCR4 agonist pepducin that mobilizes bone marrow hematopoietic cells

The G protein-coupled receptor (GPCR), chemokine CXC-type receptor 4 (CXCR4), and its ligand, CXCL12, mediate the retention of polymorphonuclear neutrophils (PMNs) and hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Agents that disrupt CXCL12-mediated chemoattraction of CXCR4-expressing cells mobilize PMNs and HSPCs into the peripheral circulation and are therapeutically useful for HSPC collection before autologous bone marrow transplantation (ABMT). Our aim was to develop unique CXCR4-targeted therapeutics using lipopeptide GPCR modulators called pepducins. A pepducin is a synthetic molecule composed of a peptide derived from the amino acid sequence of one of the intracellular (IC) loops of a target GPCR coupled to a lipid tether. We prepared and screened a small CXCR4-targeted pepducin library and identified several pepducins with in vitro agonist activity, including ATI-2341, whose peptide sequence derives from the first IC loop. ATI-2341 induced CXCR4- and G protein-dependent signaling, receptor internalization, and chemotaxis in CXCR4-expressing cells. It also induced dose-dependent peritoneal recruitment of PMNs when administered i.p. to mice. However, when administered systemically by i.v. bolus, ATI-2341 acted as a functional antagonist and dose-dependently mediated release of PMNs from the bone marrow of both mice and cynomolgus monkeys. ATI-2341-mediated release of granulocyte/macrophage progenitor cells from the bone marrow was confirmed by colony-forming assays. We conclude that ATI-2341 is a potent and efficacious mobilizer of bone marrow PMNs and HSPCs and could represent a previously undescribed therapeutic approach for the recruitment of HSPCs before ABMT.     

鼠疫菌4Mdal质粒的发现及鉴定

用琼脂糖凝胶电泳检测分离自云南省澜沧和勐海两县的鼠疫菌质粒,发现除具有已知的6、45和65三种质粒外,尚存在一种分子量约4Mdal 的新质粒。此质粒的证实对研究该地区鼠疫特征及在传播方面将具有重要的流行病学意义。     

内皮细胞固有型一氧化氮合酶 4 b/a多态性与终末期慢性肾衰竭的关联性研究

目的对天津地区汉族人内皮细胞固有型一氧化氮合酶基因内含子4的插入/缺失多态性(ecNOS 4 b/a)与终末期慢性肾衰竭 (ESCRF)的关联性进行研究.方法应用PCR-小卫星DNA多态性分析技术,对67例ESCRF患者(观察组)和70例健康人(对照组)的ecNOS 4 b/a基因型分布进行检测.结果 (1) 观察组和对照组的三种基因型bb、ba和aa频率分别为79.1%、19.4%、1.5%、91.4%、8.6% 和0% .(2)两组等位基因分布差异有显著性(χ2=4.617,P＜0.05).观察组比对照组a/(a+b)OR值(95%可信区间)为2.64(1.09, 6.43)(Z=2.14,P＜0.05).(3)有a基因的ESCRF患者平均年龄(47.43±11.63)岁,无a基因的ESCRF患者平均年龄(58.08±13.68)岁,二者比较差异有显著性(t=2.664,P＜0.01).(4) 天津汉族正常人群a基因频率(4.3%)低于日本人(10.1%)(χ2=4.898,P=0.027).结论天津地区汉族健康人群a等位基因频率低于日本人和澳洲白种人;ecNOS 4 b/a多态性与天津地区汉族人ESCRF有关联性,a基因与ESCRF危险性增加呈正相关.     

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor

Unlike conventional αβ T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1⁺ and Vδ2⁺ γδ TCR-mediated ligand recognition, the mode of Vδ3⁺ TCR ligand engagement is unknown. MHC class I–related protein, MR1, presents vitamin B metabolites to αβ T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2⁻ γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3⁺ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3⁺ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Characterized by both innate and adaptive immune cell functions, γδ T cells are an unconventional T cell subset. While the functional role of γδ T cells is yet to be fully established, they can play a central role in antimicrobial immunity (1), antitumor immunity (2), tissue homeostasis, and mucosal immunity (3). Owing to a lack of clarity on activating ligands and phenotypic markers, γδ T cells are often delineated into subsets based on the expression of T cell receptor (TCR) variable (V) δ gene usage, grouped as Vδ2⁺ or Vδ2⁻.The most abundant peripheral blood γδ T cell subset is an innate-like Vδ2⁺subset that comprises ∼1 to 10% of circulating T cells (4). These cells generally express a Vγ9 chain with a focused repertoire in fetal peripheral blood (5) that diversifies through neonatal and adult life following microbial challenge (6, 7). Indeed, these Vγ9/Vδ2⁺ T cells play a central role in antimicrobial immune response to Mycobacterium tuberculosis (8) and Plasmodium falciparum (9). Vγ9/Vδ2⁺ T cells are reactive to prenyl pyrophosphates that include isopentenyl pyrophosphate and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (8) in a butyrophilin 3A1- and BTN2A1-dependent manner (10–13). Alongside the innate-like protection of Vγ9/Vδ2⁺ cells, a Vγ9⁻ population provides adaptive-like immunobiology with clonal expansions that exhibit effector function (14).The Vδ2⁻ population encompasses the remaining γδ T cells but most notably the Vδ1⁺ and Vδ3⁺ populations. Vδ1⁺ γδ T cells are an abundant neonatal lineage that persists as the predominating subset in adult peripheral tissue including the gut and skin (15–18). Vδ1⁺ γδ T cells display potent cytokine production and respond to virally infected and cancerous cells (19). Vδ1⁺ T cells were recently shown to compose a private repertoire that diversifies, from being unfocused to a selected clonal TCR pool upon antigen exposure (20–23). Here, the identification of both Vδ1⁺ T_naive and Vδ1⁺ T_effector subsets and the Vδ1⁺ T_naive to T_effector differentiation following in vivo infection point toward an adaptive phenotype (22).The role of Vδ3⁺ γδ T cells has remained unclear, with a poor understanding of their lineage and functional role. Early insights into Vδ3⁺ γδ T cell immunobiology found infiltration of Vδ3⁺ intraepithelial lymphocytes (IEL) within the gut mucosa of celiac patients (24). More recently it was shown that although Vδ3⁺ γδ T cells represent a prominent γδ T cell component of the gut epithelia and lamina propria in control donors, notwithstanding pediatric epithelium, the expanding population of T cells in celiac disease were Vδ1⁺ (25). Although Vδ3⁺ IELs compose a notable population of gut epithelia and lamina propria T cells (∼3 to 7%), they also formed a discrete population (∼0.2%) of CD4⁻CD8⁻ T cells in peripheral blood (26). These Vδ3⁺ DN γδ T cells are postulated to be innate-like due to the expression of NKG2D, CD56, and CD161 (26). When expanded in vitro, these cells degranulated and killed cells expressing CD1d and displayed a T helper (Th) 1, Th2, and Th17 response in addition to promoting dendritic cell maturation (26). Peripheral Vδ3⁺ γδ T cells frequencies are known to increase in systemic lupus erythematosus patients (27, 28), and upon cytomegalovirus (29) and HIV infection (30), although, our knowledge of their exact role and ligands they recognize remains incomplete.The governing paradigms of antigen reactivity, activation principles, and functional roles of γδ T cells remain unresolved. This is owing partly due to a lack of knowledge of bona fide γδ T cell ligands. Presently, Vδ1⁺ γδ T cells remain the best characterized subset with antigens including Major Histocompatibility Complex (MHC)-I (31), monomorphic MHC-I–like molecules such as CD1b (32), CD1c (33), CD1d (34), and MR1 (35), as well as more diverse antigens such as endothelial protein coupled receptor (EPCR) and phycoerythrin (PE) (36, 37). The molecular determinants of this reactivity were first established for Vδ1⁺ TCRs in complex with CD1d presenting sulfatide (38) and α-galactosylceramide (α-GalCer) (34), which showed an antigen-dependent central focus on the presented lipids and docked over the antigen-binding cleft.In humans, mucosal-associated invariant T (MAIT) cells are an abundant innate-like αβ T cell subset typically characterized by a restricted TCR repertoire (39–43) and reactivity to the monomorphic molecule MR1 presenting vitamin B precursors and drug-like molecules of bacterial origin (41, 44–46). Recently, populations of atypical MR1-restricted T cells have been identified in mice and humans that utilize a more diverse TCR repertoire for MR1-recognition (42, 47, 48). Furthermore, MR1-restricted γδ T cells were identified in blood and tissues including Vδ1⁺, Vδ3⁺, and Vδ5⁺ clones (35). As seen with TRAV 1-2⁻, unconventional MAITs cells the isolated γδ T cells exhibited MR1-autoreactivity with some capacity for antigen discrimination within the responding compartment (35, 48). Structural insight into one such MR1-reactive Vδ1⁺ γδ TCR showed a down-under TCR engagement of MR1 in a manner that is thought to represent a subpopulation of MR1-reactive Vδ1⁺ T cells (35). However, biochemical evidence suggested other MR1-reactive γδ T cell clones would likely employ further unusual docking topologies for MR1 recognition (35).Here, we expanded our understanding of a discrete population of human Vδ3⁺ γδ T cells that display reactivity to MR1. We provide a molecular basis for this Vδ3⁺ γδ T cell reactivity and reveal a side-on docking for MR1 that is distinct from the previously determined Vδ1⁺ γδ TCR-MR1-Ag complex. A Vδ3⁺ γδ TCR does not form contacts with the bound MR1 antigen, and we highlight the importance of non–germ-line Vδ3 residues in driving this MR1 restriction. Accordingly, we have provided key insights into the ability of human γδ TCRs to recognize MR1 in an antigen-independent manner by contrasting mechanisms.     

Rabbit immunoglobulin kappa genes: structure of a germline b4 allotype J-C locus and evidence for several b4-related sequences in the rabbit genome.

To investigate the genetic mechanism by which certain rabbits can express immunoglobulins unexpected on the basis of their pedigree (i.e., "latent" allotypes), we have begun a study of the rabbit immunoglobulin kappa gene locus. Here we report the structure of a germline genomic clone that encodes the b4 allotype of rabbit kappa immunoglobulin and corresponds to the kappa gene expressed by the rabbit-mouse hybridoma 12F2. The nucleotide sequences of the joining (J) and constant (C) regions reveal structures generally similar to the homologous mouse and human loci, although only one of the five J-like sequences of this rabbit gene is apparently expressed. Southern blotting analysis of DNA from several rabbit allotypic strains by using probes derived from the cloned b4 gene demonstrates that, in contrast to mouse and human, rabbits possess multiple kappa-related sequences. Rabbits of the nominal b4, b5, b6, and b9 allotypes each contain at least two b4-related sequences that are associated with their own J regions and that are highly homologous to the cloned b4 gene in both coding and flanking regions.     

CXCR4 and CD4 mediate a rapid CD95-independent cell death in CD4+ T cells

AIDS is characterized by a progressive decrease of CD4⁺ helper T lymphocytes. Destruction of these cells may involve programmed cell death, apoptosis. It has previously been reported that apoptosis can be induced even in noninfected cells by HIV-1 gp120 and anti-gp120 antibodies. HIV-1 gp120 binds to T cells via CD4 and the chemokine coreceptor CXCR4 (fusin/LESTR). Therefore, we investigated whether CD4 and CXCR4 mediate gp120-induced apoptosis. We used human peripheral blood lymphocytes, malignant T cells, and CD4/CXCR4 transfectants, and found cell death induced by both cell surface receptors, CD4 and CXCR4. The induced cell death was rapid, independent of known caspases, and lacking oligonucleosomal DNA fragmentation. In addition, the death signals were not propagated via p56^lck and G_iα. However, the cells showed chromatin condensation, morphological shrinkage, membrane inversion, and reduced mitochondrial transmembrane potential indicative of apoptosis. Significantly, apoptosis was exclusively observed in CD4⁺ but not in CD8⁺ T cells, and apoptosis triggered via CXCR4 was inhibited by stromal cell-derived factor-1, the natural CXCR4 ligand. Thus, this mechanism of apoptosis might contribute to T cell depletion in AIDS and might have major implications for therapeutic intervention.     

Discovery of the recoverable high-pressure iron oxide Fe4O5

Phases of the iron-oxygen binary system are significant to most scientific disciplines, directly affecting planetary evolution, life, and technology. Iron oxides have unique electronic properties and strongly interact with the environment, particularly through redox reactions. The iron-oxygen phase diagram therefore has been among the most thoroughly investigated, yet it still holds striking findings. Here, we report the discovery of an iron oxide with formula Fe(4)O(5), synthesized at high pressure and temperature. The previously undescribed phase, stable from 5 to at least 30 GPa, is recoverable to ambient conditions. First-principles calculations confirm that the iron oxide here described is energetically more stable than FeO + Fe(3)O(4) at pressure greater than 10 GPa. The calculated lattice constants, equation of states, and atomic coordinates are in excellent agreement with experimental data, confirming the synthesis of Fe(4)O(5). Given the conditions of stability and its composition, Fe(4)O(5) is a plausible accessory mineral of the Earth's upper mantle. The phase has strong ferrimagnetic character comparable to magnetite. The ability to synthesize the material at accessible conditions and recover it at ambient conditions, along with its physical properties, suggests a potential interest in Fe(4)O(5) for technological applications.     

Polymorphism of human erythrocyte C3b/C4b receptor.

The human erythrocyte receptor for the major activation fragments of the third and fourth components of complement (HuE-C3bR) was isolated from individual donors. Erythrocytes were surface labeled with 125I and solubilized in Nonidet P-40.HuE-C3bR was purified by using C3-Sepharose affinity chromatography and analyzed by autoradiography of NaDodSO4/polyacrylamide gels. Three distinct receptor patterns were demonstrated. Type a had a single major band with Mr of 190,000, type b had a single major band with Mr of 220,000, and type c had two major bands of Mr 190,000 and 220,000. In all three types, a minor band accounting for less than 25% of the total radioactivity was usually observed at a Mr 15,000 greater than that of each major band. Identical autoradiographic patterns were obtained by affinity chromatography using methylamine-inactivated C4-Sepharose or by immunoprecipitation of solubilized membranes with a monoclonal antibody against HuE-C3bR. All three types were distinct after reduction and alkylation, although the apparent Mr uniformly increased by approximately equal to 30,000. Characterization of HuE-C3bR types in 33 unrelated individuals demonstrated that 23 had type a, 1 had type b, and 9 had type c. Family studies provide evidence for transmission by two codominant alleles. Thus, in the normal population two alleles appear to control expression of HuE-C3bR phenotypes and account for the polymorphism of this integral membrane glycoprotein.     

Discovery and Evolutionary Analysis of a Novel Bat-Borne Paramyxovirus

Paramyxoviruses are a group of RNA viruses, such as mumps virus, measles virus, Nipah virus, Hendra virus, Newcastle disease virus, and parainfluenza virus, usually transmitted by airborne droplets that are predominantly responsible for acute respiratory diseases. In this paper, we identified a novel paramyxovirus belonging to genus Jeilongvirus infecting 4/112 (3.6%) bats from two trapping sites of Hainan Province of China. In these animals, the viral RNA was detected exclusively in kidney tissues. This is the first full-length Jeilongvirus genome (18,095 nucleotides) from bats of genus Hipposideros, which exhibits a canonical genome organization and encodes SH and TM proteins. Results, based on phylogenic analysis and genetic distances, indicate that the novel paramyxovirus formed an independent lineage belonging to genus Jeilongvirus, representing, thus, a novel species. In addition, the virus-host macro-evolutionary analysis revealed that host-switching was not only a common co-phylogenetic event, but also a potential mechanism by which rats are infected by bat-origin Jeilongvirus through cross-species virus transmission, indicating a bat origin of the genus Jeilongvirus. Overall, our study broadens the viral diversity, geographical distribution, host range, and evolution of genus Jeilongvirus.     

Metabolism of c4 and factor b in rheumatoid arthritis

Metabolic turnover determined by radioiodide labeled C4 and Factor B was studied in 18 patients with rheumatoid arthritis (RA) and 19 normal control subjects as a means of estimating the relative ratio of consumption of components in the classical and alternative pathways of complement activation. Predominance of fractional catabolic rate (FCR) of C4 over Factor B was demonstrated with differentially labeled C4 and Factor B. The hypercatabolism occurred in the extravascular space. C4 FCR correlated significantly with rheumatoid factor (RF) determined in a hemolytic assay (r_s = 0.72), measured as IgG RF (r_s = 0.57), and as IgM RF (r_s = 0.45). There were no significant correlations with several other antibodies measured. These results are consistent with the hypothesis that RA is a systemic, extravascular immune complex disease, in which RF immune complexes play a significant pathogenetic role principally via activation of the classical pathway of complement.     

Discovery and functional characterization of a neomorphic PTEN mutation

Although a variety of genetic alterations have been found across cancer types, the identification and functional characterization of candidate driver genetic lesions in an individual patient and their translation into clinically actionable strategies remain major hurdles. Here, we use whole genome sequencing of a prostate cancer tumor, computational analyses, and experimental validation to identify and predict novel oncogenic activity arising from a point mutation in the phosphatase and tensin homolog (PTEN) tumor suppressor protein. We demonstrate that this mutation (p.A126G) produces an enzymatic gain-of-function in PTEN, shifting its function from a phosphoinositide (PI) 3-phosphatase to a phosphoinositide (PI) 5-phosphatase. Using cellular assays, we demonstrate that this gain-of-function activity shifts cellular phosphoinositide levels, hyperactivates the PI3K/Akt cell proliferation pathway, and exhibits increased cell migration beyond canonical PTEN loss-of-function mutants. These findings suggest that mutationally modified PTEN can actively contribute to well-defined hallmarks of cancer. Lastly, we demonstrate that these effects can be substantially mitigated through chemical PI3K inhibitors. These results demonstrate a new dysfunction paradigm for PTEN cancer biology and suggest a potential framework for the translation of genomic data into actionable clinical strategies for targeted patient therapy.The application of next generation sequencing in cancer biology has produced a deluge of genomic alterations present across human cancers (1, 2). This information has helped identify common mutational signatures in cancer types and subtypes (3–5). However, application of systematic patient-specific sequencing data to guide personalized cancer treatment has been limited in practice (6–8). The key challenge is functional understanding of the individual genetic lesions in the given phenotypic context and its translation into actionable strategies for targeted treatment.To test the utility of an unbiased computational and experimental framework in causal variant identification and characterization, we sequenced to high coverage the entirety of a prostate tumor biopsy and its matched normal (buccal) tissue from a 59-y-old Hispanic male with aggressive prostate adenocarcinoma [stage T3a; Gleason score 7 (4+3)] (SI Appendix, Fig. S1A).     

An Individual with Hb-Lepore-Baltimore- δβ-Thalassaemia in a Yugoslavian Family

The clinical, haematological and biochemical findings in a person with δβ-thalassaemia and Hb-Lepore are described. The patient was a 24-year-old student who suffered from anaemia of intermediate seventy with late onset of the clinical manifestations, had minor bone and facial deformities, but had no necessity for regular transfusions. Haemoglobins A and A₂ were absent in this individual, and the Hb-Lepore has been identified as Lepore-Baltimore. Heterogeneity of γ chain of the Hb-F follows the expected pattern. The study provides further evidence that neither β nor δ chains are synthesized in cis to δβ-thalassaemia or Hb-Lepore.     

Hypermethylation of P16ink4a and P15ink4b genes as a marker of disease in the follow-up of non-Hodgkin's lymphomas

The hypermethylation of p16ink4a and p15ink4b genes have been described as an inactivating mechanism alternative to deletions and mutations that accounts for a relatively high proportion of cancers, including non-Hodgkin's lymphomas (NHLs). To investigate whether detection of abnormal methylation could have clinical applications in the management and follow-up of lymphomas, we have analysed the behaviour and evolution of p16ink4a and p15ink4b methylation in 13 NHL cases undergoing chemotherapy. All cases were also analysed for the presence of monoclonal rearrangements of immunoglobulin or T-cell receptor genes. Six patients showed methylation in at least one of these genes at diagnosis, whereas in two other cases methylation appeared during the treatment. The other five cases were always unmethylated. Methylation was detected when any histological or molecular evidence of disease was present, suggesting a good correlation between methylation and disease. In some cases, we were able to detect methylation in patients at complete remission and without evidence of monoclonal cell population, indicating a high sensitivity of the PCR to detect methylation. These results suggest that p16ink4a and p15ink4b methylation could be good markers of disease and could be helpful in identifying lymphoma patients at risk of relapse.     

Discovery of a novel Raf kinase inhibitor

We discuss the biology of Ras signal transduction and the epidemiology of ras mutations in association with disease as a background for the development of a Raf kinase inhibitor, BAY 43-9006. Knowledge of Ras effector pathways has permitted genetic validation of numerous targets involved in the Ras signaling cascade. A key Ras effector pathway involves the kinase cascade RAF/MEK/ERK (MEK: MAP/ERK kinase; ERK: extracellular signal related kinase). Indeed, we present studies of cell lines stably expressing mutant MEK constructs, which point to Raf kinase as a target for therapeutics with selective anti-tumor activity. Finally, a small molecule drug discovery program based on inhibition of Raf kinase activity is outlined and the initial pre-clinical development process of the Raf kinase inhibitor BAY 43-9006 is discussed.